Expression and characterization of six clinically relevant uroporphyrinogen decarboxylase gene mutations.

The functional consequence of six uroporphyrinogen decarboxylase (UROD) gene mutations found in Danish patients with familial porphyria cutanea tarda was investigated. Wild-type UROD and the 6 mutants (3 missense, 1 nonsense and 2 frameshift mutants) were cloned and expressed using the prokaryotic gGEX-6P system, in which the protein is produced in fusion with glutathione S-transferase (GST). Enzymatic activity of the purified recombinant mutant fusion proteins ranged from undetectable to less than 12% of the recombinant wild-type protein. Mutant proteins cleaved from the GST part did not retain any catalytic activity. These observations can be ascribed to the structure/function relationships of the enzyme, and the fact that the enzyme is a dimer in its active form. Although the clinical manifestation of familial porphyria cutanea tarda is complex, the findings support the notion that different mutations may affect individuals differently.

Characterization of two isoalleles and three mutations in both isoforms of purified recombinant human porphobilinogen deaminase.

Defects in the enzyme porphobilinogen deaminase (PBG-D) are associated with acute intermittent porphyria (AIP). Human PBG-D is transcribed into a housekeeping or an erythroid form as a result of differential promoter usage and splicing. In addition, three pairs of isoallelic forms have been described. However, whether the enzymatic properties of housekeeping and erythroid forms differ is unknown. In this study the two isoallelic forms, K210 and E210, were cloned and expressed in Escherichia coli together with three mutations associated with a clinical AIP phenotype. The mutations were introduced in the K210 isoallelic background and expressed as both the housekeeping and the erythroid form. The proteins were expressed as GST fusions and purified to homogeneity. Initial experiments revealed that the GST-PBG-D fusions and the purified PBG-D obtained by proteolytic removal of the GST moiety had enzymatic properties that were indistinguishable. Consequently, all analyses with mutant PBG-D were performed on the GST-fusion proteins. Comparison of the wild-type proteins revealed a significant difference in Km between isoalleles with a Km of 9 microM for K210 and 7 microM for E210, whereas no significant difference in activity or kinetics between the housekeeping and the erythroid isoforms was observed. The mutant proteins showed 0.3-1.0% wild-type activity, depending on mutation. There was a clear correlation between yield of recombinant protein and CRIM status of patients. Furthermore, co-expression of the mutant proteins with the bacterial chaperone GroESL did not affect protein yield or function to any significant extent, supporting the view that the investigated mutations primarily influence structure and function and not folding of the proteins.

Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis. Identification and characterization of six novel mutations associated with familial PCT.

The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able to detect the underlying UROD mutation in 10 previously characterized DNA samples as well as a new mutation in each of six previously unexamined PCT patients. The six novel UROD mutations comprise three missense mutations (M01T, F229L, and M324T), two splice mutations (IVS3-2A-->T and IVS5-2A-->G) leading to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual catalytic activity, whereas the two missense mutants retained some residual activity. Furthermore, the missense mutants exhibited a considerable increase in thermolability. The six new mutations bring to a total of 29 the number of disease-related mutations in the UROD gene. The DGGE assay presented greatly improves the genetic diagnosis of fPCT and HEP, thereby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases.

An improved PCR-based method for site directed mutagenesis using megaprimers.

An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.